Engineered sers substrates employing nanoparticle cluster arrays with multiscale signal enhancement

ABSTRACT

Defined nanoparticle cluster arrays (NCAs) with dimensions up to 25.4 μm square are fabricated on a 10 nm gold film using template guided self-assembly. Structural parameters are precisely controlled, allowing systematic variation of the number of nanoparticles in the clusters (n) and edge to edge separation (Λ) between 1&lt;n&lt;20 and 50 nm≦Λ≦1000 nm, respectively. Rayleigh scattering spectra and surface enhanced Raman scattering (SERS) signal intensities as functions of n and Λ reveal direct near-field coupling between the particles within individual clusters, whose strength increases with cluster size (n) until it saturates at around n=4. Strong near-field interactions between clusters significantly affects the SERS signal enhancement for edge-to-edge separations Λ&lt;200 nm. The NCAs support multiscale signal enhancement from simultaneous inter- and intra-cluster coupling and |E|-field enhancement. Applications include SERS-based spectral identification of bacteria.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Contract No.W911NF-06-2-0040 awarded by the Army Research Office. The Government hascertain rights in the invention.

BACKGROUND

The intensities and frequencies of vibrational transitions measured inRaman spectra provide unique chemical signatures of molecular species,but the sensitivity of Raman spectroscopy suffers from the relativelylow cross-section of inelastically scattered Raman photons. It has beendemonstrated that the magnitude of Raman cross-sections can be greatlyenhanced when the Raman-active molecules are placed on or near aroughened noble metal surface. Since then a wide variety of substrateshave been found to enable surface enhanced Raman spectroscopy (SERS)such as aggregated noble metal colloids, metal island films, metal filmover nanospheres, particles grafted on silanized glasses, regular holesin thin noble metal films and regular nanoparticle arrays.

In general, traditional SERS substrates can be divided into twofundamental substrate classes: random and engineered substrates. Randomsubstrates like fractal nanoparticle agglomerates can support localizeddipole modes which lead to high SERS signal enhancements. However, theresonance wavelength, the precise locations of the spots of giant|E|-field enhancement—so called hot-spots—and the reproducibility oftheir enhancement factors are difficult to control in completely randomstructures. Another disadvantage specific to fractal nanoparticleaggregates is that their mass density, and therefore the hot-spotdensity, decreases with increasing fractal size.

Challenging applications of SERS in single molecule spectroscopy orwhole organism fingerprinting would greatly benefit from engineered SERSsubstrates with rational design criteria that generate high SERSenhancement reproducibly at spatially defined locations. Consequently,regular nanoparticle arrays and other nanofabricated SERS substrates,whose characteristic structural parameters can be accurately controlled,are attracting interest as SERS substrates with reproducible, highenhancement factors “by design”. The SERS enhancement in noble metalnanoparticle arrays depends on both the properties of the constitutivebuilding blocks (nanoparticles) as well as the characteristics of theirarrangement. In general two separate electromagnetic regimes govern thecollective response of periodic metal-nanoparticle arrays: near andfar-field coupling. When the particles are separated by short distancesup to approximately D=1/k₀=λ₀/2π (with k₀ and λ₀ being the free spacewavenumber and wavelength, respectively), strong quasi-static near-fieldinteractions dominate the response of the array. Consequently, localizedmodes with strongly enhanced local fields are excited. When theparticles are separated by larger distances, far-field diffractivecoupling between the particles becomes dominant.

In the near-field coupling regime the field enhancement andcorresponding SERS intensity arising from periodic arrays ofnanoparticles sharply increases with decreasing inter-particleseparation. Both theoretical and experimental studies have shown thatregions of high |E|-field enhancement are located in the junctionbetween individual particles. The |E|-field enhancement in thesespatially confined hot spots can be orders of magnitude larger than onthe surface of individual particles. Due to the rapid decay of the fieldstrength with inter-particle separation and the |E|⁴ scaling of the SERSsignal, very short inter-particle separations are vital in order tomaximize the Raman enhancement in the near-field coupling regime.Ideally, the analyte molecules are placed in the junctions betweennearly touching metal surfaces.

SUMMARY

It remains challenging to create junction plasmons at predefinedlocations and with nanometer accuracy in current top-down fabricationmethods such as electron beam (e-beam) lithography. The spatialresolution of e-beam lithography is limited by laterally scatteredsecondary electrons, which makes it difficult to reproducibly fabricatearrays with inter-particle separations of less than 10 nm. In order toovercome this limitation, an alternative approach is demonstrated herethat can be used to engineer SERS substrates with nanoscaleinter-particle separations reliably. Template guided chemicalself-assembly is used to create nanoparticle cluster arrays (NCAs) ofdefined size with nanoscale inter-particle separations at predefinedpattern locations. E-beam lithography is not used to directly generateplasmonic structures but instead to define binding sites on whichchemically synthesized gold nanoparticles can assemble. Consequently,“hot” inter-nanoparticle junctions at predefined locations in a regulararray can be created, enabling the possibility to control and optimizeboth near- and far-field noble metal nanoparticle interactions.

The description herein includes a systematic characterization of theoptical scattering spectra and the Raman signal intensity enhancementsof these NCAs as function of cluster size n and cluster edge-to-edgeseparation Λ, comparing their performance with non-patterned colloidalgold films and periodic two-dimensional nanodisc arrays, and anapplication for spectral identification of bacterial pathogens. Rapidbacteria diagnostics are vital for improving the treatment outcomes ofserious infections and ensuring the appropriate use of antibioticstrategies. A SERS based approach for bacterial detection andidentification relies on signal amplification techniques (PCR), and thusoffers several potential advantages, such as speed, reducedsusceptibility to contamination problems, ease-of-use and mixtureresolution for rapid, specific and sensitive bacterial diagnostics. Thekey requirement for the success of this methodology is the production ofSERS substrates with large and reproducible signal enhancement. In thisstudy, we demonstrate that NCAs provide reproducible SERS signals fromdifferent bacteria species including Escherichia coli, Bacillus cereus,and Staphylococcus aureus.

Overview of Methods

Nanofabrication of Particle Binding Sites.

A fabrication process is described which begins with spin-coating 180 nmpolymethyl methacrylate (PMMA) 950 photoresist on top of Au-coated (10nm Au film) glass slides. The substrates are subsequently soft-baked at180° C. for 20 min. Periodic patterns of nanowells are then written witha Zeiss SUPRA 40VP SEM equipped with Raith beam blanker and ananopattern generation system (NPGS). After e-beam writing, thephotoresist is developed in methyl isobutyl ketone (MIBK). Periodicpatterns of nanowells with inter-well separations (edge to edge) of 50nm, 100 nm, 150 nm, 200 nm, 400 nm, 600 nm, 800 nm, and 1000 nm aregenerated by this procedure.

Template Guided Self-Assembly of Gold Nanoparticle Clusters.

Commercial citrate-stabilized 40 nm Au particles in aqueous solution areconcentrated by a factor of 10 by centrifugation. 100 μL of theconcentrated gold sol is then incubated with 5 μL of a 10 mMthiol-EG₇-propionat (EG=ethylene glycol) aqueous solution overnight atroom temperature. The particles are cleaned by centrifugation andre-suspended in a 10 mM phosphate buffer pH=8.6 containing 40 mM NaCl.The patterned gold substrates are incubated with a 1 mM aqueous solutionof thiol-(CH2)₁₁EG₇-Amine for 15 minutes and then washed with water. TheAu particles solution is added on the top surface of the substrates andincubated for 1 h. The particle solution is removed by washing withwater. After the samples are dried, PMMA liftoff is performed with1-Methyl-2-pyrroldinone.

Dark-Field Scattering Characterization of Periodic Cluster Arrays.

Scattering images of the particle cluster arrays were recorded using anupright microscope (Olympus BX51 WI). The nanoparticle arrays wereimmersed in index-matching oil (n_(r)=1.5) and illuminated withunpolarized white-light from a 100W tungsten halogen lamp using an oildark-field condenser (NA 1.2-1.4) in transmission mode. The lightscattered from the arrays was collected with a 60× oil immersionobjective (NA=0.65) and imaged using a digital camera with an activearea of 620×580 pixels. The microscope was also equipped with a 150 mmfocal length imaging spectrometer (Acton Research, InSpectrum 150) and aback-illuminated CCD detector (Hamamatsu INS-122B) that enabled thespectral analysis of the scattered light using a 150 lines/mm grating.The scattering spectra were background corrected by subtraction of thescattering signal from an equal-size, non-patterned adjacent area. Thescattering spectra were additionally corrected by the excitation profileof the white light source by normalizing with the scattering spectrum ofan ideal white light scatterer on top of the gold film.

SERS Measurements.

A Renishaw Raman microscope (model RM-2000) capable of ˜2λ spatialresolution was used to observe the scattering excited by a 785 nm diodelaser. The frequency calibration was set by reference to the 520 cm-1silicon phonon mode. Paramercaptoaniline (pMA) was used to characterizethe field enhancement on the cluster arrays. The saturated aqueous pMAsolution was kept on the substrate for 10 min before removing with aflow of nitrogen gas. A 50× objective (numerical aperture NA=0.78) wasused for signal collection. SERS spectra were acquired with incidentlaser powers in the 0.44 to 7.34 mW range and acquisition times of 10-60seconds.

Calculation of Approximate SERS Enhancement Factors forParamercaptoaniline (pMA).

SERS enhancement factors, G, were calculated following standardprocedures G is defined here by:G=(ISubstrate/NSubstrate)*(NReference/IReference) where ISubstrate isthe Raman intensity of a monolayer of pMA on the SERS substrate andIReference is the Raman signal due to a pMA crystal. NReference andNSubstrate refer to the number of pMA molecules in a monolayer on theSERS substrate and in the focal region of the crystal, respectively. Anaperture was used to confine the sample detection area to 6=2.5 μm×25μm. NSubstrate was obtained as the ratio of active nanoparticle clusterarray area within the detection area and the cross-section of the pMAmolecule (σ_(pMA)=0.3 nm²). For NCAs the active area is estimated bymultiplying the number of clusters in the laser spot with the product ofthe average number of particles in the clusters and the surface area ofone hemisphere of a 40 nm gold nanoparticle. NReference was calculatedassuming a confocal depth of 14 μm and a density of 1.06 g/mL for solidpMA (molecular weight=125 g/mol). For non-patterned colloidal gold filmsthe surface-densities of 40 nm gold particles were obtained by countingthe number of particles in representative SEM images with defineddimensions. Then NSubstrate was calculated using the same assumptions asin the case of the NCAs. In the case of smooth nanodisc arrays,NSubstrate was determined as the ratio of active area (number of discsin the detection area multiplied by the exposed disc surface area) andσ_(pMA).

Bacteria Growth and Sample Preparation.

Gram-negative bacteria Escherichia coli (ATCC #12435), and Gram-positivebacteria Bacillus cereus (ATCC #14579) and Staphylococcus aureus (ATCC#25904) were grown in 15-20 mL of LB (Sigma) for ˜5 h at 37° C. untilthey reached an OD600=˜0.6. About 4 mL of each culture solution waswashed, centrifuged and vortexed four times with Millipore water.Finally, the pellet was suspended in 0.25 mL of water. About 1 μL of thebacteria suspension was placed on the cluster arrays, and after thewater had evaporated (˜2 minutes), the samples were transferred into theRaman microscope to record SERS spectra.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIGS. 1 and 2 are a top view and side view respectively of a substratefor use in SERS spectroscopy;

FIGS. 3 and 4 are magnified top and side views respectively of thesubstrate of FIGS. 1 and 2;

FIGS. 5 and 6 are sets of scanning electron microscope (SEM) images of atop surface of a SERS substrate;

FIG. 7 is a plot of normalized SERS scattering spectra for variousvalues of inter-cluster separation;

FIG. 8 is a plot of peak resonance wavelength as a function ofinter-cluster separation;

FIG. 9 is a plot of cluster size distribution as a function of size ofbinding site;

FIG. 10 is a plot of peak wavelength as a function of cluster size,along with an inset showing corresponding Rayleigh scattering spectra;

FIG. 11 is a set of diagrams illustrating a method and correspondingworkpieces in fabricating a SERS substrate;

FIG. 12 is a diagram depicting binding of a gold nanoparticle to abinding site;

FIG. 13 is a plot of SERS spectra of a monolayer of pMA for variousvalues of inter-cluster separation;

FIG. 14 is a plot of SERS enhancement factors for a monolayer of pMA asa function of inter-cluster separation;

FIG. 15 is a plot of SERS intensity for a monolayer of pMA as a functionof binding site size;

FIG. 16 is a plot of SERS enhancement factor for a monolayer of pMA as afunction of cluster size;

FIG. 17 is a plot showing a comparison of SERS enhancement factors fordifferent types of SERS substrates;

FIG. 18 is a plot showing Rayleigh scattering spectra for the differenttypes of SERS substrates of FIG. 17;

FIG. 19 is a plot showing SERS spectra of three different bacteria S.aureus, E. coli, and B. cereus;

FIG. 20 is a discriminant function analysis (DFA) plot derived from thedata of FIG. 19;

FIG. 21 is a plot of SERS spectra of a monolayer of pMA for variousvalues of laser excitation power; and

FIG. 22 is a plot of SERS signal strength as a function of integrationtime.

DETAILED DESCRIPTION

FIGS. 1 and 2 show top and side views respectively of a substrate 10 foruse in SERS spectroscopy in the form of a glass slide 12 having apatterned top surface 14 used as the active SERS surface. FIG. 3illustrates a close-up view of the top surface 14, on which is formed anarray of clusters 16 of gold nanoparticles. FIG. 4 shows a set ofadjacent clusters 16 in side view. The schematic side view of FIG. 4shows nanoparticles 18 of each cluster 16. FIG. 4 also shows dimensionsD and Λ, with D being the diameter of the binding sites at which eachcluster 16 is located and Λ being an edge-to-edge separation of theclusters 16. The term “cluster size” is used herein to refer to thenumber of nanoparticles 18 per cluster 16. It will be appreciated thatthe cluster size is a function of both the dimension D and the diameterof a nanoparticle 18.

FIG. 5 shows scanning electron microscope (SEM) images of sections ofthe fabricated cluster arrays 16 with a fixed edge-to-edge separation(Λ=200 nm) but varying binding site diameters (D=50 nm, 80 nm, 100 nm,130 nm, 200 nm). The SEM images show regular arrays of two-dimensionalnanoparticle clusters with varying cluster size. The images confirm thattemplate guided self-assembly approach (described below) leads to aspatially confined particle clustering into discrete assemblies. FIG. 5also shows that the average cluster size (i.e. number of particles inthe clusters) can be conveniently controlled by varying the binding sitediameter D. For D=50, regular arrays of individual 40 nm goldnanoparticles with a high degree of translational symmetry are found.With increasing D, larger clusters are formed that exhibit some geometryand size variability. Even under ideal assembly conditions adistribution of particle numbers and cluster geometry results from thenatural size dispersion of the colloid used (coefficient of variation<20%). The low number of particles in the clusters makes the variationin the cluster size and shape most striking for intermediate bindingsizes of D=80 nm to 130 nm. In contrast, the larger clusters appear morehomogenous again.

Image (f) of FIG. 5 shows a particle cluster in the D=80 nm NCA athigher magnification. As evident the particle clusters contain holes,junctions, and crevices between a few nanometer-spaced goldnanoparticles. These nanoparticle-gap structures are known to support astrong |E|-field localization which can induce a strong enhancement ofthe dipole moment of the analyte molecules. The enhanced moleculardipole moments and the amplification of the re-radiated Raman scatteredlight through the noble metal nanostructures lead to a strongenhancement of the SERS signal.

To test the field enhancing effect of inter-cluster coupling, NCAs weremade with varying cluster edge-to edge separation Λ. NCAs with Λ=50,100, 150, 200, 400, 600, 800, and 1000 nm and fixed binding sitediameters between D=50-200 nm were made. Of these, FIG. 6 shows exampleswith fixed binding site diameter D of 200 nm and separations Λ of 50 nm,200 nm, and 1000 nm. It may be difficult to decrease the edge-to-edgeseparation significantly below Λ=50 nm, especially for larger clustersizes, because at very short inter-particle separations individualnanoparticles clusters tend to fuse and form continuous lines ofnanoparticle clusters across the pattern. Thus, the current templateassisted self-assembly approach can generate extended NCAs with a widerange of inter-cluster separation Λ and cluster sizes D, albeit withsome potential limitation for the minimum inter-cluster separation. Thefollowing description presents a systematically analysis of the opticalresponse and the SERS enhancement as functions of Λ and D to elucidatean understanding the fundamental mechanisms governing the multi-lengthscale electrodynamic interactions in NCAs.

Influence of the Cluster Edge-to-Edge Separation Λ on the RayleighScattering Spectra of Nanocluster Particle Arrays.

The optical response of dielectrically coated gold nanoparticles on agold film is determined by the local plasmons of the particles, theirinteractions through space (near-field and far-field coupling) and bytheir coupling to delocalized plasmon modes supported by the gold film.The localized modes in the particles can couple with image modes in thegold film as well as with plasmon modes in neighboring particles. Inaddition, a gold surface which is periodically corrugated by goldnanoparticle clusters can act as a grating coupler. In this case photonsincident on this surface can efficiently excite a propagating surfaceplasmon in the gold film, which can be Bragg-scattered from the regularcluster arrays.

Given these different electromagnetic interactions between localized anddelocalized plasmon modes in NCAs, it is challenging to quantitativelypredict the optical response of the nanoparticle cluster arrays. As afirst step towards an understanding of these potential SERS substratesthe spectral response of the NCAs can be characterized as a function ofthe controllable template parameters Λ and D. To that end arrays may befabricated with varying cluster edge-to-edge separations Λ=50-1000 nmbut constant cluster binding sizes D=200 nm. Since we kept the totalnumber of binding sites constant for all inter-cluster separations D,the total area of the fabricated arrays varies from 25.4 μm×25.4 μm forΛ≦1000 nm to 16 μm×16 μm for Λ=50 nm. Due to the small size of thearrays a spectral characterization of the arrays using extinctionspectroscopy would be challenging. Instead, we decided to characterizethe optical response of the NCAs using Rayleigh scattering spectroscopy,which can be conveniently performed in a darkfield microscope. With thehelp of a darkfield condenser the excitation light can be injected intothe specimen plane at such a steep angle from the bottom that onlyscattered light can reach the objective on top of the sample. Thegeometrical constraints of the darkfield illumination allows effectivediscrimination of the excitation light, and is therefore an idealtechnique for probing the plasmon resonances of nanostructures which donot provide a strong extinction. Rayleigh scattering spectroscopy isroutinely used to investigate the optical properties of a wide range ofnanostructured plasmonic materials, ranging from single noble metalnanoparticles over regular one- and two-dimensional arrays of goldnanoparticles to deterministic aperiodic arrays of gold nanoparticles.

FIG. 7 shows the normalized scattering spectra of the fabricated NCAswith varying Λ but fixed D=200 nm. The displayed spectral intensitieswere not corrected by the different filling fractions of the arrays,since we focus on the spectral shifts originating from differences inthe array parameters in this study. All of the spectra in FIG. 7 show abroad peak around 700 nm, which we assign to the nanoparticle clusterresonance coupled via propagating surface plasmons in the gold supportas has been described elsewhere. In addition to this couplednanoparticle cluster resonance, NCAs with Λ<200 nm exhibit a separateshort wavelength band which strongly red-shifts with increasing Λ. InFIG. 8, the peak resonance wavelengths of these two bands, determined byGaussian best fits, are plotted as a function of Λ. The maximum of theshort wavelength band shifts from ˜455 nm (Λ=50 nm) over ˜545 nm (Λ=100nm) to ˜613 nm (Λ=150 nm). For Λ≧200 nm a separate second peak can nolonger be resolved. However, the spectrum of the NCA with Λ=200 nm (FIG.7) is asymmetrically broadened on the high energy side, indicating thepossibility of additional spectral features.

Short wavelength bands next to the plasmon of the nanostructures havebeen observed previously in extinction measurements of regular arrays ofsmooth nanodiscs on gold and were successfully ascribed to propagatingsurface plasmons or standing waves due to Bragg scattering at thenanoparticle array on top of the gold film. These models have, however,only limited applicability in our scattering analysis of NCAs becausethe investigated array geometries do not fulfill the grating couplingconditions at our illumination angle (see below) in the investigatedwavelength range; in addition the correspondence between calculatedBragg scattering resonances and the measured high energy is approximateat best. Due to the experimental geometry of the performed scatteringexperiments an alternative explanation for the short wavelength bandsarises from the possibility that the periodic two-dimensional structureof gold nanoparticle clusters on top of a transparent 10 nm thin goldfilm acts as a transmission grating for some components of the incidentlight. Diffraction of the wavelength λ incident at angle φ_(inc) on theoil immersed NCA is then described by the grating formula:

L(sin φ_(inc)+sin θ_(det))=(m/n _(r))λ  (1)

where L is cluster center-to-center separation, θ_(det) is the detectionangle, m is the diffraction order, and n_(r) is the refractive index ofglass and index matching oil (n_(r)=1.5).

The incident angle φ_(inc) is determined by the numerical aperture ofthe darkfield condenser. For a numerical aperture of NA=1.2,φ_(inc)=53°. The maximum detection angle θ_(det) is given by themarginal ray as determined by the objective numerical aperture. Thenumerical aperture of the objective used in these studies (NA=0.65)results in a maximum θ_(det)≦25.7°. All wavelengths that fulfillequation (1) for 0°<θ_(det)≦25.7° can be folded into the scatteringspectrum. Following this model and considering the emission onset of theTungsten lamp at ˜400 nm we can assign peaks in the following spectralregions to diffraction at the grating: λ=400 nm-462 nm (Λ=50 nm, L=250nm); λ=400 nm-555 nm (Λ=100 nm, L=300); λ=419 nm-647 nm (Λ=150 nm,L=350); λ=479 nm-754 nm (Λ=200 nm, L=400 nm). For larger Λ thediffracted wavelengths are shifted out of the detection range. Theexperimentally observed short wavelength bands all fall in thewavelength ranges predicted by equation (1). We conclude that the simpletransmission diffraction grating model suffices to explain the observeddependence of the high energy band on Λ.

The frequency of the lower energy band, assigned to the cluster plasmonresonance, appears to depend weakly on the cluster edge-to-edgeseparation Λ as shown in FIG. 8. This effect is, however, much smallerthan for the peak arising from diffraction in the investigated Λ range.The cluster resonance peak (red line) slightly blue shifts from 720 nmfor Λ=50 nm separations to 690 nm for Λ=200 nm structures. At largeredge-to-edge distances the cluster plasmon resonance peak does notappear to shift further. This small blue-shift with increasinginter-particle separation is attributed to direct near-fieldinteractions between the clusters of the arrays on the gold substrate.

Influence of Cluster Size on the Rayleigh Scattering Spectra ofNanoparticle Cluster Arrays.

As evidenced in FIG. 5, the average number of particles within theindividual building blocks of NCAs can be systematically tuned bychanging the diameter (D) of the binding site defined by e-beamlithography. The cluster size distribution and average cluster size n asa function of the binding size D for arrays with fixed edge-to-edgeseparation Λ=200 nm are quantitatively described in FIG. 9. For D=50 nmindividual 40 nm gold nanoparticles are the predominant building block.However, the number of particles on the binding sites grows with D, thesite diameter. The average cluster size n increases steadily from n=1.3for D=50 nm to n=4.4 for D=120 nm and then jumps to n=19.1 for D=200 nm(see FIG. 9).

The fitted peak wavelengths of the scattering spectra recorded from theNCAs analyzed in FIG. 9 are plotted as function of n in FIG. 10. Theoriginal spectra are included as inset. The peak wavelength (λ_(res))strongly red-shifts from 558 nm for n=1.3 to 670 nm for n=4.4. However,further increases of the cluster size beyond n=4.4 lead to only smalladditional redshifts of the scattering band. The peak wavelengthincreases only by 9 nm to λ_(res)=679 between n=4.4 and n=19.1.

The strong red-shift of the spectral response with increasing degree ofparticle clustering is a direct consequence of near-field couplingbetween the particles in the clusters, driven by the increasing numberof interstitial junction plasmons as n increases. Plasmon hybridizationbetween adjacent particle plasmons leads to coupled cluster resonancesthat are energetically stabilized with regard to the isolated particleplasmons. FIG. 10 shows that this stabilization saturates in largerclusters. The major portion of the energy stabilization is reached foran average cluster size of n=4.4. A further increase in the size of theclusters results in only a small additional shift of their plasmonresonance wavelength.

The red-shift due to plasmon hybridization stabilizing at around n≈4 maybe understood from simple geometric considerations. A close inspectionof the SEM images of the fabricated NCAs reveals that the clusters withn=4 preferentially assume a rhombohedral geometry in which theinter-particle distances are minimized (see FIG. 5 image (f)). Four goldnanoparticles at the edges of a rhombus form the unit cell of amonolayer of hexagonal closed packed spheres. This highly symmetricarrangement enables the particles to minimize the total inter-particleseparation resulting in very efficient plasmon coupling. The fact thatan increase in the average cluster size beyond n=4 does not lead tosubstantial additional spectral shifts in regular arrays thereforeimplies that the coupling in the larger clusters is dominated byinter-particle coupling between nearest neighbors in the unit cell.

In three-dimensional clusters additional interactions between particlesalong the third spatial axis (out-of-plane) can shift the plasmonresonance further into the red than observed here for two-dimensionalclusters. In fact, some of the fabricated NCAs contain contaminationswith larger three dimensional agglomerates due to imperfections duringthe fabrication process. Shoulders at wavelengths>800 nm in thescattering spectra in FIG. 10 are ascribed to these three-dimensionalclusters and fractal particle assemblies. The occasional contaminationof the fabricated NCAs does, however, not influence the observed globaltrends: the peak wavelength red-shifts in the interval 1<n<4 andconverges to its maximum at n≈4.

Assembly of Regular Nanoparticle Cluster Arrays.

FIG. 11 illustrates a template guided self-assembly process to generateregular two-dimensional arrays of contiguous nanoparticles. In the firststep a photoresist (PMMA) film is formed on a glass substrate covered bya thin noble metal layer (e.g., 10 nm gold film). A regular structure ofwells or openings is then created in the PMMA film using e-beamlithography. The resulting PMMA mask covers all parts of the surfaceexcept for the anticipated binding sites. The binding sites arefunctionalized by assembly of a monolayer of amino-terminated PEGs(thiol-(CH2)₁₁EG₇-amine, EG=ethylene glycol) on the exposed goldsurface. Under appropriate buffer conditions (pH<9) the monolayer ispositively charged. Thus, negatively charged 40 nm colloidal goldnanoparticles readily bind on these positively charged binding sites inan electrostatic guided self-assembly process. The gold nanoparticlesare passivated with a monolayer of carboxy terminated PEGs(thiol-EG₇-propionat, see FIG. 12). The charged polymers on the goldsurfaces serve two purposes; they facilitate an efficient chargedirected cluster assembly on the template and function as insulatingdielectric between the particles and between the nanoparticles and thegold support.

SERS Performance of Nanoparticle Cluster Arrays.

Fabrication procedures that provide spatial control on the nanoscale areinstrumental in developing SERS substrates according to rational designcriteria. Our motivation for assembling arrays of clusters of nearlytouching gold nanoparticles at defined locations is to reproduciblycreate hot-spots with high surface density to generate SERS substrateswith high enhancement factors and improved enhancement reproducibility.The spectral characterization of the fabricated cluster arrays hasalready indicated the existence of both inter-particle and inter-clusterplasmon coupling in NCAs. In order to be able to utilize the interplayof electromagnetic interactions between individual nanoparticles in theclusters and between clusters, it is important to characterize theinfluence of the array specific geometry parameters Λ and n on therelative Raman intensities. All SERS spectra in this study were excitedat 785 nm. This wavelength has been shown to minimize autofluorescencefrom biological samples.

Influence of the Cluster Edge-to-Edge Separation Λ on the SERS Signal.

Following other SERS studies, paramercaptoaniline (pMA) was used here asa test analyte to quantify the influence of Λ on the NCA Raman signalenhancement. To optimize the SERS performance of the substrates, theSERS signal dependence on pump power P (P=0.4 mW-7.3 mW) and dataacquisition time t (t=10-60 seconds) was investigated first (see FIGS.21 and 22). The pMA SERS intensity on a Λ=200 nm, D=200 nm NCA was foundto be linearly dependent on P and t. SERS substrates with good/excellentsignal to noise are obtained with 10 seconds of data collection andP=1.8 mW. All SERS measurements subsequently reported were performedwith these acquisition parameters unless otherwise stated.

FIG. 13 contains pMA SERS spectra from NCAs with different values of Λbut with fixed nanoparticle binding sizes (D=200 nm). Two effectscontribute to the strong increase of the SERS intensity with decreasingedge-to-edge separation. Firstly, the density of the nanoparticleclusters and therefore the total SERS active area in the scatteringvolume increases with decreasing Λ. Secondly, the NCA scattering spectraas function of inter-cluster separation (FIG. 8) reveals that for shortedge-to-edge separations Λ<200 nm additional inter-cluster couplingfurther enhances the local |E|-field. Such a local field enhancementeffect is evident from a comparison of SERS enhancement factors, G, asfunction of Λ. The absolute values of these enhancement factors are onlyapproximates since the surface coverage with pMA, the accessible surfacearea of the clusters, the contribution of the gold substrate, as well asthe number of molecules in the reference sample have to be estimated(see Methods section). Nevertheless, they facilitate a quantitativecomparison of the relative SERS performances of samples with differentfilling fractions (i.e. different A), prepared under otherwise identicalconditions. The approximate enhancement factors obtained for the 1077cm⁻¹ band of pMA on NCAs with constant binding site diameter (D=200 nm)are plotted as function of Λ in FIG. 14.

The measured SERS enhancement decreases from 2.2·10⁵ for Λ=50 nm to1·10⁵ for Λ=200 nm. For even larger edge-to-edge separations the SERSenhancement is essentially independent of Λ. The gain in G at shortinter-cluster separations is in agreement with the observed spectralred-shift of the plasmon resonance and is consistent with increasingnear-field interactions between the clusters for Λ<200 nm. Thenear-field inter-cluster interactions increase the SERS enhancementgenerated by individual clusters by a factor of ˜2 with respect to theisolated clusters at the smallest Λ tested in this study. Thisobservation further corroborates that plasmon coupling in NCAs occurs ontwo relevant length scales: inter-particle in the clusters andinter-cluster in the arrays.

Influence of Cluster Size on the SERS Signal.

The SERS enhancement as a function of the binding size diameter D, andthus the average cluster size, n, in NCAs with fixed edge-to-edgeseparation Λ have also been investigated.

In FIG. 15 the SERS intensities of the pMA 1077 cm⁻¹ transition of NCAswith five different D values (50 nm, 80 nm, 100 nm, 130 nm, 200 nm) andfixed edge-to-edge separation (Λ=200 nm) are compared. All of thesenanoparticle cluster arrays were fabricated on the same chip to minimizeintensity effects due to pMA concentration or pump power variability.The recorded signal intensity increases nearly linearly with growing D.It is a priori unclear how much a potential cluster size dependence ofthe SERS enhancement contributes to the signal intensity gain. Theinfluence of D on the SERS enhancement was therefore estimated bycalculating the SERS enhancement factors G as function of Λ (see FIG.16). The general trend in FIG. 16 indicates that G does not continuouslyincrease with cluster size n but converges against a maximum enhancementat n≈3-4. In the case of NCAs with fixed Λ=200 nm a maximum enhancementfactor of G≠1·10⁻⁵ is reached. This behavior corroborates the trendsobserved for the plasmon resonance wavelength.

The stagnation of the resonance wavelength at n≈4 in FIG. 10 isrationalized by a maximization of the inter-particle near-fieldinteractions in compact cluster geometries. In addition, in clusters ofthree or four particles with triangular or rhombal cluster geometry allparticles can be arranged in a “first coordination shell” around acentral cavity. Analyte molecules located in this space can potentiallyexperience very high local fields leading to strong SERS enhancementshighlighting the value of the first interstitial coordination shell forSERS signal enhancement.

Benchmarking NCAs with Competing SERS Substrates UsingParamercaptoaniline (pMA) as Test Substance.

Performance of NCAs was evaluated by direct comparison with two commonlyused SERS substrates: (1) non-patterned 40 nm gold nanoparticle filmsand (2) periodic two-dimensional arrays of gold nanodisc arrays. Thenon-patterned colloid films were conveniently generated on the samesubstrate next to the nanoparticle cluster arrays by simply removing thephotoresist from a large area in the vicinity of the surface patternduring the e-beam writing step. All subsequent processing steps wereidentical to the procedures described above for the production of thenanocluster particle arrays. The nanodisc arrays were fabricated on a 10nm thin gold film following standard procedures. In short, a PMMA maskwas generated on top of a gold coated glass slide using direct writinge-beam lithography with subsequent development. Then a 40 nm thick goldlayer was thermally evaporated onto the patterned surface and the PMMAmask was removed in a final lift-off step releasing the regular goldnanodisc pattern. The NCAs used for the benchmarking had an edge-to-edgeseparation of Λ=200 nm and a cluster binding size of D=200 nm. Thenanofabricated nanodisc arrays had the same edge-to-edge distance andtotal diameter as the NCAs.

The relative SERS performance of these three different substrates wasevaluated in direct comparison under identical conditions. The Ramanenhancement (G) factors at 785 nm for the 1077 cm⁻¹ mode of pMA andcorresponding representative SEM images of the investigated substratesare summarized in FIG. 17. These values correspond to twelvemeasurements from three different substrates for each substrate type.The NCA substrates yielded the overall largest G values (˜1.1·10⁵),followed by the non-patterned nanoparticle film (˜7.0·10⁴). The nanodiscarray exhibited the smallest G values (˜1.0·10⁴). NCAs with Λ=50 nm,D=200 nm yielded the maximal enhancement factor of G=2.2·10⁵ for thesubstrates prepared for this study (see FIG. 14). These values fallwithin the typical SERS enhancement range of gold island films and arecomparable with those obtained with other engineered SERS substratessuch as gold nanohole arrays. Although enhancement factors up to 1·10⁸have been observed in some cases for e-beam patterned gold nanodiscarrays fabricated on a gold film, the NCAs are found to provide asignificantly higher (˜1 order of magnitude) Raman signal enhancementthan the corresponding nanodisc arrays at least for pMA excited at 785nm.

The observation that the NCAs provide a stronger SERS signal enhancementthan smooth nanodisc arrays is readily ascribed to the fact that theNCAs have a much higher degree of roughness than the nanodiscs due tocrevices, holes, and junctions between the nanoparticles in theclusters. The incident field can be effectively localized in thesenano-roughened structures leading to overall higher enhancement factors.Nanoscale roughness cannot, however, account for the fact that theensemble averaged SERS enhancement factors for NCAs are also slightlyhigher than those of the non-patterned gold nanoparticle films whichalso contain nanoparticle clusters. Based on the observed dependenciesof the ensemble averaged G factors on Λ and n, we propose the followingmodel to account for the observed differences between patterned andnon-patterned colloid substrates: the SERS enhancement factors ofindividual two dimensional nanoparticle clusters saturates at aroundn≈4. In an array of patterned clusters the total SERS enhancement of theindividual clusters can be further increased through cumulativeelectrodynamic interactions occurring on two different length scales. Onthe length scale of a few tens of nanometers (inter-cluster lengthscale) the cluster plasmons couple and provide a first stage of strongenhancement of the incident electric field. This enhanced field is thenfurther increased by the intra-cluster coupling between the individualparticles of the clusters. The phenomenon therefore consists of asequential enhancement similar to the effects observed in RF Yagiantennas or optical nanolenses.

As discussed elsewhere, the Raman signal enhancement can be maximized bymatching the excitation wavelength with the absorption band of theplasmonic SERS substrate. FIG. 18 shows the normalized spectra of theinvestigated NCAs, smooth gold nanodisc arrays, and non-patternedcolloid films. Whereas NCAs and nanodisc arrays show well definedplasmon resonances at around ˜700 nm with a full width at half maximum(FWHM) extending from 630 nm to 780 nm, the non-patterned colloid filmshows a much broader scattering spectrum with FWHM between 580 nm and880 nm. We ascribe this spectral broadening to a wide distribution ofcluster sizes including three-dimensional and fractal aggregates in thenon-patterned colloid film.

The measured relative Rayleigh scattering intensities for NCAs andnanodisc arrays have decreased to ˜30% of their peak intensity value atthe SERS excitation wavelength of 785 nm. In contrast, for thenon-patterned colloid substrate the scattering cross-section is close toits peak value at the 785 nm SERS excitation wavelength. The detuningbetween 785 nm Raman excitation wavelength and the resonance scatteringmaxima evident in FIG. 18 suggests that—unlike the non-patterned colloidfilms—the enhancement factors of NCAs can be significantly increased byimproving the match between resonance wavelength and excitation laserwavelength. This can be achieved either by using a laser with anemission wavelength around 700 nm or by shifting the resonancewavelength of the arrays closer to 785 nm by exchanging spherical 40 nmgold nanoparticles with building blocks that have energetically lowerparticle resonances.

Another important SERS performance characteristic, in addition to thesignal enhancement, is the reproducibility of the enhancement factorsgenerated by different SERS substrates. The reproducibility of theenhancement factor (G) is captured here by the coefficient ofvariability, i.e. the standard deviation of G values given by twelveindependent measurements on four different substrates, expressed as apercentage of the mean G value for each substrate type. Thesevariability coefficients are given in FIG. 17. As evident from thismeasure for G reproducibility, the variations of the NCAs enhancement(12%) is much smaller than that observed on either the non-patternedcolloid substrate (41%), or the nanodiscs (56%). Thus both in terms ofabsolute enhancement and repeatability, the NCAs outperform the othertwo substrate types tested here.

SERS Bacterial Detection and Identification Using NCAs.

The SERS performance of NCAs makes them potentially useful candidatesfor complex sensing applications such as whole cell fingerprinting.There is currently significant interest in developing SERS for the rapidcharacterization and identification of bacterial pathogens. Due to thedistance dependence of the field enhancement, SERS selectively probesthe molecular components of the outer layer of bacterial cells wherechemical distinctions appear to be the greatest thus enhancingspecificity and may therefore be a promising tool for bacterialdiagnostics. The successful application of SERS for this analyticalapplication requires substrates that can provide strong and reproducibleenhancements for these organisms at the single cell level and have astorable shelf life in the 6-12 month range.

In the case of bacteria the surface morphology and the binding affinityto the substrate are extremely important and can influence both thedetected vibrational bands and the total signal intensities. Not allSERS active substrates provide SERS spectra of whole bacterial cells.Only if a bacterial cell can effectively attach to the surface such thatcharacteristic surface moieties are near SERS active sites will a strongRaman signal be observed.

In order to test the ability of the NCAs, the non-patterned colloidfilms, and the nanodiscs to act as effective substrates for theobservation of SERS spectra of vegetative bacterial cells, suspensionsof three different bacterial species (Staphylococcus aureus, Escherichiacoli, and Bacillus cereus) were placed on these substrates and SERSspectra excited at 785 nm (4.3 mW power, 10 seconds integration time)were acquired. Only very weak bacterial SERS spectra could be observedon the nanodisc arrays. However, both patterned (NCA) and non-patternedcolloid substrates provided quantifiable SERS signals. FIG. 19 showsrepresentative SERS spectra (single scan) of S. aureus, E. coli, and B.cereus obtained from NCA and smooth (40 nm height) nanodisc arrays, bothwith 200 nm diameter and 200 nm edge-to-edge separation features. Thenon-patterned gold nanoparticle substrates were located on the same chipin close vicinity to the nanoparticle cluster array to ensure that thesetwo samples were always measured under identical experimentalconditions. As seen in FIG. 19, SERS spectra of bacteria are stronger onthe NCA substrates than on the non-patterned colloid films. Furthermore,in contrast to the NCA bacterial spectra, the bacterial SERS signalintensities exhibited strong variations at different positions on thenon-patterned substrates.

The bacterial SERS spectra share many common spectral features asevident in FIG. 19 and has been discussed previously. Identification ofthe chemical species responsible for the vibrational bands in the SERSspectra of bacteria has not yet been achieved and is beyond the scope ofthis current report. However, the unique SERS vibrational signaturesprovide the basis for a rapid bacterial identification methodology whencombined with multivariate library searching techniques.

The ability of NCAs to be used for bacterial diagnostics is demonstratedin FIG. 20. It has been shown previously how a principal componentanalysis (PCA) based on the sign of the second derivative of the SERSspectra provides improved specificity for the identification ofbacterial species and strains. The SERS spectra are thus reduced to aseries of zeroes and ones, i.e. barcodes, as input for unsupervisedclustering algorithms such as PCA, or consequent supervised methods suchas discriminant function analysis, hierarchical cluster analysis, orneural network techniques. Here the specificity of these SERS basedsignatures on NCAs is shown by the results of a discriminant functionanalysis (DFA) based on the barcode reduced SERS spectra of S. aureus,E. coli, and B. cereus. The discriminant functions are linearcombinations of the first four PCs which capture 98% of the variance ofthis 30 spectra data set.

As seen in the DF2 vs. DF1 plot in FIG. 20, the SERS signature of B.cereus, E. coli, and S. aureus obtained on the NCAs are well separatedforming non-overlapped regions. The rings in each of the cluster regionscorrespond to tow dimensional standard deviations centered on the meanfor each species cluster. The large standard deviation separationsbetween these clusters of the tested clusters of the tested bacteriaindicate that the NCAs enable a spectral signature capable of bacterialidentification. Work is currently ongoing to determine the bestperforming SERS substrates for this purpose based on the criteria ofRaman enhancement strength, spectral reproducibility, substrate storagelifetime and commercial scalability.

It is shown that nanoparticle cluster arrays (NCAs) provide reproducibleSERS signals from different bacteria species including Bacillus cereus,Escherichia coli, and Staphylococcus aureus. The NCAs enabled aspectroscopic discrimination of these samples through SERS incombination with multivariate data analysis techniques. The NCAs usedfor this analytical challenge were fabricated by combining top downnanofabrication and bottom-up self assembly procedures in a templateguided self-assembly process. This approach provides control over thesize of the particle clusters and their spatial location on thenanoscale. We used this process to fabricate regular arrays of 40 nmgold nanoparticle clusters of defined cluster size n and clusteredge-to-edge separation Λ over several hundred square microns. Thephotonic-plasmonic scattering resonances of the arrays as function of nand Λ were characterized. The spectra are dominated by the ensembleresonance of the gold film supported nanoparticle clusters at largecluster separations. For NCAs with short inter-cluster separations,Λ<200 nm, we also observe an additional short wavelength band which weascribe to light diffraction from the NCAs acting as transmissiongrating for the incident light. A systematic variation of Λ revealedthat the plasmon resonance peak red-shifts with decreasing Λ foredge-to-edge separations Λ≦200 nm indicating additional inter-clusternear-field interactions. The red-shift of the plasmon resonance isaccompanied by an increase in the SERS enhancement for Λ≦200 nm. Thisobservation confirms that electrodynamic interactions between theclusters can further increase the Raman signal intensity generated byindividual isolated clusters, and we conclude that the net enhancementis the result of a multiscale field enhancement in NCAs. Next to theedge-to-edge separation, the SERS signal enhancement also depends on thecluster size n, and we investigated the optical response and the SERSenhancement of NCAs as function of n. The cluster resonances of thearrays strongly red-shift with increasing cluster size n up to n≈4. Wedid not observe a further significant increase in the enhancement forlarger two-dimensional nanoparticle clusters. Similarly, the Ramansignal enhancement shows a significant increase with growing clustersize for small cluster sizes n≈4 but remains essentially constant forlarger cluster sizes. This behavior suggests that for n=4 clusterstructures are accessible which enable very efficient plasmon couplingbetween all particles of the clusters. We find that one of the preferredstructures for n=4 is the rhombus which is the unit cell of atwo-dimensional close packing. The size dependency of the SERSenhancement indicates a dominance of the interstitial first shell in theRaman signal amplification.

Overall, it is revealed that NCAs can be used to engineer SERSsubstrates whose spectral and field localization properties can becontrolled systematically by varying n and Λ. We benchmarked NCAs withnon-patterned two-dimensional gold nanoparticle substrates and regulargold nanodisc arrays. We found that NCAs offer a good compromise betweensignal enhancement and substrate reproducibility. In addition, the NCAsclearly outperformed the other substrates in SERS measurements ofbacteria. Future steps for further improvement and optimization of theSERS enhancement by NCAs will involve the study of the nanoparticlecomposition, size, and shape as well as the geometric patterns of thearrays.

While various embodiments of the invention have been particularly shownand described, it will be understood by those skilled in the art thatvarious changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

What is claimed is:
 1. A substrate for use in surface-enhanced Ramanspectroscopy, comprising: a planar substrate layer; and a nanoparticlecluster array on a surface of the planar substrate layer, thenanoparticle cluster array including an array of clusters of metalnanoparticles characterized by a cluster size n and a cluster separationΛ, n being a nominal number less than 10 of tightly-packed nanoparticlesin each cluster determined by a nominal size of the nanoparticles and adeterministic binding site width D, and Λ being a deterministic distanceless than 200 nm between adjacent clusters.
 2. A substrate according toclaim 1, wherein n is determined by the radius of the nanoparticles andmorphology and width D of the binding sites.
 3. A substrate according toclaim 2, wherein the nanoparticles are made of a material selected fromsilver, copper, silver-gold alloys, and aluminum.
 4. A substrateaccording to claim 2, wherein the nanoparticles shapes are selected fromnon-spherical and/or hollow, core-shell nanoparticles and multi-scaleaggregates of different size/shape nanoparticles.
 5. A substrateaccording to claim 2, wherein the nanoparticles are mixed dielectric andmetallic structures.
 6. A substrate according to claim 1, wherein thesubstrate layer includes a functionalizing first monolayer, and thenanoparticles include a functionalizing second monolayer bound to thefirst monolayer at the binding sites.
 7. A substrate according to claim1, wherein the substrate is a non-conducting dielectric material.
 8. Asubstrate according to claim 1, wherein the substrate contains aconducting film on a non-conducting dielectric.
 9. A substrate accordingto claim 1, wherein the substrate is a metal.
 10. A substrate accordingto claim 1, wherein the substrate is a flexible conducting ornon-conducting polymer.
 11. A substrate according to claim 1, whereinthe nanoparticles are from metal nitrides or germanides.